Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

Abstract

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl.

Figure 1.

(A) Proposed mechanism of Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE). Chromosomal mutations are introduced via single-stranded oligonucleotide recombination on the lagging strand of replicating DNA, mediated by the β subunit of λ Red recombinase. Under ordinary conditions (left), the newly-synthesized strand is hemimethylated, which enables the MMR system, composed of MutS, MutL and MutH, to bind to mismatched lesions and restore the wild-type allele. In standard MAGE (middle), the MMR system is eliminated, allowing incorporation of the mismatched base and its complement base into the genome of one daughter cell after a second round of replication (red). However, permanent disabling of mismatch repair also allows the accumulation of off-target mutations (yellow) during subsequent manipulations. In TM-MAGE (right), transient overexpression of DNA adenine methylase (Dam) is hypothesized to generate significant methylation of the newly-synthesized strand, disabling detection of incorporated mismatches by the MMR system and reducing the rate of off-target mutations that accumulate in subsequent manipulations as Dam expression returns to its baseline level. (B) Schematic of TM-MAGE system plasmids pMA7 (top) and pMA7SacB (bottom). Plasmid pMA7 contains an artificial operon of the gene encoding the β subunit of λ Red recombinase and dam controlled by an arabinose-inducible (P_BAD) promoter. Plasmid pMA7SacB additionally contains constitutively expressed sacB (levansucrase from B. subtilis) to allow for plasmid curing by sucrose counterselection.

Figure 2.

Allelic replacement efficiency (percent recombinants) with various lengths of Dam induction in K-12 MG1655/pMA7 prior to chilling cells for electrocompetent cell preparation. Ten minutes was found to result in optimal allelic replacement frequencies (ARFs) following a single cycle of MAGE with an oligonucleotide introducing a premature stop codon in galK (Y145*). ARFs were determined by counting red (wild-type allele) and white (mutant allele) colonies appearing following plating on MacConkey agar containing 1% galactose.

Figure 3.

Transient expression of Dam enables efficient recombineering. ARFs for xylA Y13* in strains EcNR2, K-12 MG1655/pMA7 and K-12 MG1655/pMA7SacB following one cycle of MAGE with a 90 bp phosphorothioated oligonucleotide. ARFs were determined both by counting red (wild-type allele) and white (mutant allele) colonies appearing following plating on MacConkey agar containing 1% xylose, and by amplicon sequencing within the xylA locus. Error bars indicated standard deviations about the mean of three replicates (two for K-12 MG1655/pMA7SacB).

Figure 4.

ARF from TM-MAGE with pMA7 are nearly equivalent to those obtained when mismatch repair is permanently disabled. ARFs for galK Y145* (top) and xylA Y13* (bottom) using 70-bp non-modified oligonucleotides were determined by amplicon sequencing using cell pellets obtained following each of 6 cycles of MAGE in strains K-12 MG1655/pMA1, K-12 MG1655/pMA7 and K-12 MG1655 mutS::cat/pMA1. ARFs were also determined from counting red (wild-type allele) and white (mutant allele) colonies appearing following plating after 6 cycles of MAGE on MacConkey agar containing galactose or xylose. Measurements were performed on single replicates.

Figure 5.

Spontaneous mutation rates are elevated only when Dam overexpression is induced, and are reduced compared to permanently disabled MMR. (A) Frequencies of rifampicin and nalidixic acid resistant mutants following plating of non-induced strains (K-12 MG1655 with and without pMA1, K-12 MG1655 mutS::cat with and without pMA1 and K-12 MG1655 with pMA7). (B) Frequencies of rifampicin and nalidixic acid resistant mutants arising following plating of induced strains that had undergone a procedure mimicking a single MAGE cycle. Individual replicate measurements are shown, with the median value denoted by a horizontal black line.

Figure 6.

Population frequencies of mutant alleles (indicated at right) as determined by amplicon sequencing following 6 cycles of MAGE and after 1, 3 and 5 days of daily passaging in M9 medium or M9 medium plus NaCl (0.5 M for the first passage and 0.6 M for subsequent passages). Libraries were generated in K-12 MG1655/pMA7 (black bars), K-12 MG1655/pMA1 (gray bars) and K-12 MG1655 mutS::cat/pMA1 (white bars). Error bars indicate standard deviations about the mean values of three biological replicate cultures. Only a single replicate was sequenced following 6 cycles of MAGE, therefore, no error bars are shown.

Figure 7.

Strains with the highest growth rates and greatly reduced lag times were reliably isolated from NaCl-selected libraries generated using TM-MAGE, as compared to other MAGE systems. (A–C) Averaged growth curves across three biological replicate cultures of control strains and individual isolates (A: K-12 MG1655/pMA7, B: K-12 MG1655/pMA1, C: K-12 MG1655 mutS::cat/pMA1) following 5 days of passaging on M9 + NaCl. (D) Calculated growth rates and (E) lag times of starting strains and isolated strains following M9 + NaCl selections. Error bars represent standard deviations about the mean of three biological replicate measurements.